The major objectives of this proposal are: a) to prepare a variety of bidentate sulfonamide inhibitors of zinc(II) metalloproteins, and b) develop a cell-based screening method for examining the effect of these inhibitors on several metalloenzymes simultaneously. Matrix metalloproteinases (MMPs) play a central role in the progression of inflammatory disease, stroke, arthritis, cancer, and heart disease. All MMPs utilize a zinc(II) ion to perform the catalytic hydrolysis of connective tissue and other biological substrates. The overall aim of this project is to develop improved matrix metalloproteinase inhibitors (MMPi) that utilize zinc(II)-specific sulfonamide chelators as novel zinc-binding groups (ZBGs). Chelating sulfonamides will enhance binding to the zinc(II) metalloprotein active site, while sparing other metalloenzymes (e.g. iron- dependent enzymes). To validate this hypothesis, the selectivity of these inhibitors for MMPs will be assessed using a novel macrophage-based screening methodology. The goals of this proposal will be achieved via the following specific aims: 1. The synthesis and characterization of simple, chelating sulfonamide ZBGs. Combined with bioinorganic model complexes that reproduce the MMP active site, sulfonamide ZBGs will provide essential information on the binding mode and affinity of these ligands for the zinc(II) ion. 2. The design and synthesis of novel MMPi. Using a combined bioinorganic-computational approach from our laboratory, potent and selective MMPi will be devised based on new sulfonamide ZBGs. 3. The evaluation of novel MMPi using fluorescence and colorimetric assays. New MMPi will be evaluated against a variety of recombinant MMPs. 4. The screening of MMPi against several metalloenzymes in a macrophage cell model of inflammation. MMPi will be examined in a novel macrophage cell-based assay that will determine the selectivity of the inhibitors for MMPs versus other zinc(II)-dependent metalloenzymes, heme- iron metalloenzymes, and non-heme-iron metalloenzymes. By using this approach and achieving these aims, we will identify new MMPi with greater affinity, selectivity, and biocompatibility than current MMPi, and thereby identify inhibitors that will be clinically successful. The success of this project will generate at least two highly innovative and high-impact findings: a) the first time that the use of zinc(II)-selective chelating sulfonamides, which are frequently found in zinc(II) ion molecular sensors, will be utilized for zinc(II)-dependent metalloprotein inhibition, and b) the development of a cell-based screening method for metalloprotein inhibitors that evaluates the effect of a compound against a broad set of metal-dependent enzymes in a biological setting. The development of drugs that can selectively inhibit metal-dependent enzymes is critical to the treatment of illnesses such as stroke, arthritis, cancer, and heart disease. This project seeks to discover selective metalloprotein inhibitors and develop a facile method by which the selectivity of these inhibitors can be rapidly assessed. PUBLIC HEALTH RELEVANCE: The development of drugs that can selectively inhibit metal-dependent enzymes is critical to the treatment of illnesses such as stroke, arthritis, cancer, and heart disease. This project seeks to discover selective metalloprotein inhibitors and develop a facile method by which the selectivity of these inhibitors can be rapidly assessed.